GPCR homodimers: structure-function studies Accumulating evidence suggests that GPCRs are able to form dimers and/or higher-order oligomeric complexes. Class A receptors represent by far the largest subfamily of GPCRs, consisting of 670 members in humans. Several studies suggest that class A receptor homodimers/oligomers are endowed with functional properties that differ from the monomeric receptor species. A proper understanding of how class A GPCRs function at the molecular level requires the identification of the structural elements governing the dimerization/oligomerization of this class of receptors. To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M3 muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. Functional assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (50% reduction in receptor-G protein coupling, as compared to the control M3R). These findings indicate that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Since all family A GPCRs share a high degree of structural homology, our data are likely to be of broad general relevance. High-resolution X-ray structure of the M3 muscarinic acetylcholine receptor (M3R) Unfortunately, no high-resolution structural information is currently available for any of the five muscarinic receptor subtypes. Given the many important physiological functions mediated by the M3R subtype, we, in collaboration with Dr. Brian Kobilka's group at Stanford University, focused on obtaining an X-ray structure of this receptor. We recently succeeded in solving the structure of the rat M3R bound to the bronchodilator drug, tiotropium, an inverse muscarinic agonist that is widely used clinically for the treatment of chronic obstructive pulmonary disease (COPD). This structure clearly identifies all amino acids that form the ligand binding pocket. These residues are located on the inner surfaces of several TM helices and are 100% conserved among all five muscarinic receptor subtypes, explaining why it has been so difficult to develop subtype-selective muscarinic ligands targeted at this so-called orthosteric binding site. The M3R structure also reveals several features that appear to be specific for muscarinic receptors, including a large extracellular vestibule, which is less well conserved among the five muscarinic receptors, and a bend at the top of TM4. Moreover, the cytoplasmic side of the M3R displays distinct structural features that are probably related to its ability to selectively interact with G proteins of the Gq family. It is likely that the high-resolution M3R structure will facilitate the rational design of new, M3R subtype-selective muscarinic drugs. Moreover, since all five muscarinic receptor subtypes share a high degree of sequence conservation, this new structural information should be highly relevant for the entire muscarinic receptor family.